This proposal is for an R21, Role of cell tropism for pathogenesis and vector competence of Zika virus. The project is a collaboration between Drs. Gregory Ebel (Colorado State University) and Aaron Brault (Centers for Disease Control and Prevention). The overall goal of this research is to understand how different cells contribute to sexual and mosquito transmission of Zika. Our overall approach to this project involves using a newly developed cDNA clone of the PRABC59 strain of Zika virus (from a Puerto Rican patient, 2015) that has been engineered to express miRNA target sequences. These sequences restrict virus replication in organism- tissue- and cell-specific manners due to binding of the cognate miRNA to viral RNA and subsequent degradation of the vRNA by the RNAi pathway. Aim 1 focuses on developing appropriate constructs and evaluating efficacy using cell lines and primary cultures. Aim 2 moves the project into an in vivo testing phase using Ag129 mice, which are available at CDC as a breeding colony, and Aedes aegypti mosquitoes, which are available at CSU as several newly established colonies. The proposed research leverages extensive preliminary data and a developing collaboration that brings together expertise in molecular virology, arboviral pathogenesis and vector biology.